Many infectious organisms increase the internal cyclic AMP (cAMP) levels of infected host cells. Whereas, some do this through chemical modification of heterotrimeric G-proteins, at least four bacteria, Bordetella pertussis (Whooping cough), Pseudomonas aeruginosa (various nosocomial infections), Yersinia pestis (plague) and Bacillus anthracis (Anthrax) accomplish this by producing toxins with adenylyl cyclase activity (Leppla, 1982; Ladant and Ullmann, 1999; Yahr et al., 1998; Shevchenko and Mishankin, 1987; Parkhill et al., 2001; Michankin et al., 1992).
Bacillus anthracis, the bacterium responsible for anthrax is estimated to be responsible for 20,000-100,000 cases of naturally contracted anthrax per year, worldwide. Anthrax may cause death within 24 hours, preceded only by nondescript, flu-like symptoms (Hanna 1998). In part, because the spore form of the bacterium is easily stored for long periods, anthrax has undergone significant development as a biological weapon.
Bacillus anthracis defeats the host defense system by secreting three exotoxins, protective antigen, lethal factor and Edema Factor (EF) (Baillie and Read, 2001; Dixon et al., 1999; Mock and Fouet, 2001). Protective antigen, a pH-dependent transporter, binds and transports both lethal factor and Edema Factor into the cytosol of eukaryotic cells. Lethal factor, a zinc metalloprotease, cleave and inactivate mitogen-activated protein kinase kinase and possibly other cellular proteins in the macrophage (Duesbery et al., 1998). This results in the release of tumor necrosis factor a and interleukin-1 b which are partly responsible for sudden death in systemic anthrax. Edema Factor, a calmodulin (CaM)-activated adenylyl cyclase, can increase the level of intracellular cyclic AMP to a pathologic level, upsetting water homeostasis (Baillie and Read, 2001; Dixon et al., 1999; Mock and Fouet, 2001). EF is responsible for the massive Edema seen in cutaneous anthrax and impaired neutrophil function in systematic infection.
Edema Factor is a 92.5 kDa protein that can be roughly divided into two distinct domains (Leppla, 1998). The N-terminal 250 amino-acid region of EF is centrally involved in EF's association with protective antigen and entrance into host cells (Labruyere et al., 1990, Drum et al. 2000). The C-terminal, 510 amino acid region (amino acids 291-800) has calmodulin activated adenylyl cyclase activity (Labruyere et al., 1990, Drum et al. 2000). After gaining access to the cytoplasm, Edema Factor binds calmodulin at the resting calcium concentration and is thus, activated to produce unregulated levels of cAMP within the cytosol of the host cell (Leppla, 1984; Leppla, 1982). The rate of ATP conversion to cAMP by Edema Factor is two to three orders of magnitude greater than membrane-bound eukaryotic adenylyl cyclases (Labruyure et al, 1990; Tang et al., 1991).
Calmodulin is a 16.5 kDa prototypic calcium sensor involved in modulation of many intracellular processes including synaptic plasticity, control of gene transcription, ion conductivities, vesicular fusion, and learning (Deisseroth et al., 1998; DiAntonio, 2000; Ehlers and Augustine, 1999; Peters and Mayer, 1998). Calmodulin transduces intracellular calcium signals via two globular domains connected by a flexible, central a-helix. Each globular domain is composed of two helix-loop-helix “EF-hand” calcium binding motifs (Babu et al., 1985; Chattopadhyaya et al., 1992; Wilson and Brunger, 2000). Calcium binding shifts these domains from a mainly hydrophilic, closed state, to an open conformation exposing a large, hydrophobic binding pocket (Kuboniwa et al., 1995; Finn et al., 1995). An exceptionally broad array of effector molecules are regulated through interaction with CaM, including enzymes that control diffuisible messengers and that alter the state of protein phosphorylation, molecules that modulate ion conductivity, and cytoskeletal proteins (Eldik and Watterson, 1998). Although the structure of CaM has been solved in complex with an assortment of effector fragments, none of these complexes has contained active enzymes or peptide fragments larger than 10 kDa.
The structural characterization of the adenylyl cyclase domain of EF holds promise for insight in several different areas. First, secreted adenylyl cyclases compose a multigene family of exotoxins produced by at least four distinct pathogenic bacteria, Bacillus anthracis, Bordetella pertussis, Pseudomonas aeruginosa and Yersinia pestis. The structural features peculiar to this homologous family may provide insight into overall functional themes of toxin activation and inhibition. Second, the catalytic rate of EF is approximately one hundred times faster than that of membrane bound adenylyl cyclases whose catalytic core domain has been structurally determined (Tesmer et al., 1997). With no sequence homology detectable between the two adenylyl cyclase families, a structural understanding of the active site of EF may yield a novel fold used for the cyclization of ATP or a variant on the di-metal mediated catalysis used by membrane-bound adenylyl cyclases (Tesmer et al., 1999). Third, the mechanism of activation with which the binding of calmodulin regulates the production of cyclic AMP in some pathogenic bacteria may hold implications for inhibitory strategies of the toxin as well as structural themes applicable to other calmodulin binding enzymes.
The inventors recently reported biochemical characterization of EF/CaM interaction (Drum et al., 2000). Drum et al. provided a detailed look of the interaction between CaM and EF, and suggested a catalytic mechanism for converting ATP to cAMP that is different from that seen in previously solved adenylyl cyclases (Tesmer et al., 1999). To understand the molecular detail in the catalytic site of EF and the interaction of EF with CaM, the inventors have solved two structures; the structure of EF without CaM or substrate, and the structure of EF with CaM, but without substrate. Comparison of these structures, along with mutational analysis reveals that the key step in CaM activation of EF is the removal of an inhibitory domain which is distant from the active site and the stabilization of a catalytic loop. Such understanding of this mechanism not only gives a first molecular insight into how CaM activates EF, but also provides potential avenues for modulating CaM activation of EF.
The present invention discloses these structures and also discloses various methods of screening assays to identify small molecules that modulate activation of EF by CaM and EF alone.
Adenylyl cyclase toxins are known to be secreted by two other human pathogens, Bordetella pertussis (CyaA) and Pseudomonas aeruginosa (ExoY) which cause whooping cough and 10-20% of hospital-acquired infections, respectively (Ladant and Ullmann, 1999; Yahr et al, 1998). Immuno-compromised patients, such as, cystic fibrosis and AIDS patients, are particularly vulnerable. Since these pathogens can be hospital acquired, they are likely to harbor many antibiotic resistance. Thus, many antibiotics can be ineffective in fighting against such infections. Accordingly, better drugs against these pathogens will likely be useful to block the toxic effects caused by the infection of these pathogens.
Also, biochemical studies have shown that Yersinia pestis, the bacterium that causes plague, also secretes an adenylyl cyclase toxin and genome sequence identified a gene in Yersinia pestis for such a toxin (Shevchenko and Mishankin, 1987; Parkhill et al., 2001; Michankin et al., 1992). Yersinia pestis is primarily a rodent pathogen which is usually transmitted subcutaneously to humans by the bite of an infected flea.
The structure of Edema Factor discovered by the inventors can be useful in discovering small molecules that can be useful against the infection of Bacillus anthracis, Bordetella pertussis, Pseudomonas aeruginosa and Yersinia pestis. 